Use of designer GPCRs to study GPCR regulation of key metabolic pathways Armbruster et al. (PNAS 104, 5163-8, 2007) first described a set of muscarinic receptor-based designer GPCRs which are now generally referred to as DREADDs ('designer receptors exclusively activated by designer drugs'). These designer receptors are unable to bind the endogenous muscarinic receptor agonist, acetylcholine, due to two single point mutations introduced into the transmembrane receptor core. Importantly, DREADDs can be efficiently activated by a compound called clozapine-N-oxide (CNO), an agent that is otherwise pharmacologically inert. The first DREADDs that were developed represent GPCRs that selectively activate G proteins of the Gq or Gi family, respectively. We recently generated additional DREADDs endowed with different coupling properties. For example, we designed an M3 muscarinic receptor (M3R)/beta1-adrenergic receptor hybrid DREADD that is able to selectively activate Gs (Guettier et al., PNAS 106, 19197-202, 2009). We also generated an M3R-based DREADD that is unable to couple to G proteins but retains arrestin-dependent signaling (Nakajima and Wess, Mol Pharmacol 82, 575-82, 2012). Analogously, we recently designed an M3R-based DREADD that shows the opposite coupling profile: lack of arrestin recruitment but efficient coupling to Gq-type G proteins (Hu X and Wess J, unpublished results). We are currently in the process of expressing DREADDs with different coupling properties in metabolically relevant cell types. These cell types include adipocytes, pancreatic beta-cells, skeletal muscle cells, and hepatocytes. Preliminary results indicate that CNO treatment of some of these mutant mouse strains has pronounced effect on glucose and energy homeostasis (unpublished results). We are also using flex switch technology to selectively express various DREADDs in distinct neuronal subpopulations of the hypothalamus, including agouti-related peptide (AgRP)-expressing neurons located in the arcuate nucleus of the hypothalamus. These neurons are known to play a key role in triggering appetite. Using flex switch technology, we recently expressed a Gs-coupled designer GPCR (Gs DREADD) selectively in AgRP neurons. CNO-dependent activation of the Gs DREADD resulted in a significant increase in food intake that continued for several days. By using different experimental approaches, we demonstrated that this effect was mediated by the release of AgRP and that NPY and GABA, which are also released from AgRP neurons, do not make a major contribution to the observed orexigenic effect. We also identified the molecular pathway through which activation of the Gs-DREADD promotes AgRP release. We speculate that agents able to inhibit signaling via Gs-coupled GPCRs endogenously expressed by AgRP neurons could prove useful to suppress appetite for therapeutic purposes (Nakajima K and Wess J; unpublished results). For recent reviews, see: Rossi M, Cui Z, Nakajima K, Hu J, Zhu L, Wess J. Virus-mediated expression of DREADDs for in vivo metabolic studies. Methods Mol Biol. 2015;1335:205-21. Nakajima K, Gimenea LE, Gurevich VV, Wess J. Design and analysis of an arrestin-biased DREADD. In: Designer Receptors Exclusively Activated by Designer Drugs, ed. G. Thiel, Springer Science + Business Media, New York, NY, in press.